PROJECT SUMMARY/ABSTRACT The Flow Cytometry and Cell Sorting Shared Resource Facility (FCCS SRF) facilitates high impact cancer science within the Markey Cancer Center (MCC) and is aided by current CCSG funding. The FCCS SRF has been in continuous operation at the University of Kentucky (UK) since 1983 and under the direction of Donald Cohen, PhD, since 1992. Although an institutional core facility at UK, during the current period of CCSG funding, 68% of users were MCC members, and 53% of users were peer-reviewed funded MCC members. To support research efforts of MCC members, the FCCS SRF provides state-of-art flow cytometric services and comprehensive immune monitoring services to support cancer research by all 4 Research Programs. A variety of flow cytometric services, including multi-parametric cell phenotyping (up to 12 biomarkers simultaneously), allows users to analyze surface and intracellular biomarkers, DNA content, cell cycle, apoptosis and necrosis, cytokine/growth factor synthesis, signal transduction and cell activation. Under guidance by MCC leadership, the FCCS SRF recently expanded services to provide MCC members comprehensive immune monitoring for human basic and clinical cancer research. Immune monitoring services include processing fresh blood and tumor tissue for isolation of immune and non-immune cells for functional assay and cryopreservation and banking of cells for later analysis. Banking services are provided in collaboration with the Biospecimen Procurement and Translational Pathology SRF and the Cancer Research Informatics SRF. Freshly isolated and banked cell samples can be analyzed for a number of cellular functions including synthesis of cytokines/growth factors/other soluble factors by ELISA, ELISPOT and Luminex multiplexing, in addition to flow cytometric approaches. Assays are also offered to quantify antigen-specific T cell proliferation, cytotoxicity, antibody synthesis and cytokine production in human systems. Cellular product services include generation of dendritic cells and macrophages from blood and/or tumor tissue and expansion of T cells from blood and/or tumor tissue. In collaboration with the Biostatistics and Bioinformatics SRF, FCCS offers high dimensional data analysis for multi-parameter flow cytometry and Luminex multi-analyte profiling for identification of complex cell populations and rare cell subsets. Over the previous CCSG funding period, FCCS services have contributed to 118 publications, 49 grants funded during the last four years, and has initiated consultation and collaboration on 4 clinical trials by MCC members. The comprehensive flow cytometry and immune monitoring services offered by FCCS, in combination with FCCS immune assay design services, are providing MCC researchers with the exceptional opportunities to interrogate novel cancer biomarkers and the immune system interactions in studies involving basic and clinical cancer research at the MCC.